Sesame (Sesamum indicum) is an annual plant in the family Pedaliaceae belonging to the genus Sesamum. Sesame is said to be indigenous to Central Africa. Sesame is supposedly the oldest domesticated oil seed crop having about a 6000 year history and has been cultivated throughout the world. Sesame is a valuable food from ancient times and known to represent healthy foods. Sesame seeds, oil pressed from sesame seeds and extracts from sesame seeds are particularly utilized (see, e.g., Goma: SONO-KAGAKU-TO-KINOSEI (Sesame: Science and Function), edited by Mitsuo Namiki, Maruzen Planet Publishing Co. (1998)). The components contained in sesame seeds are about 50% lipids and about 20% proteins. The major components of lipids contained in sesame are triglycerides mainly composed of oleic acid and linoleic acid. Furthermore, sesame contains vitamins B1, B2, E, etc. In addition to the components described above, secondary metabolites (e.g., sesamin, sesamolin, etc.) of plants collectively referred to as lignans are contained in sesame, and these components have potent anti-oxidative properties (see, e.g., Biochemical Systematics and Ecology, 13, 133-139 (1985)).
As to biosynthesis of lignans, reference is made to, e.g., Lignans: Biosynthesis and Function, Comprehensive Natural Products Chemistry, 1: 640-713 (1999); Phytochemistry Rev., 2257-288 (2003), etc.
For example, Phytochemistry Rev., 2257-288 (2003) discloses that pinoresinol synthesized through polymerization of coniferyl alcohol is the first lignan in the biosynthetic pathway and from pinoresinol a wide variety of lignans are synthesized via biosynthetic pathways inherent to individual plant species. It is reported that dirigent proteins involved in synthesis of this pinoresinol are localized in Forsythia intermedia, etc. (see, e.g., Science, 275, 362-366 (1997), etc.). In addition, pinoresinol-lariciresinol reductases genes from Forsythia intermedia (see, e.g., J. Biol. Chem., 271: 29473 (1996), Japanese National Publication (Tokuhyo) No. 2001-507931, etc.), pinoresinol-lariciresinol genes from Thuja plicata (see, e.g., J. Biol. Chem., 274: 618 (1999), etc.) as well as recombinant secoisolariciresinol dehydrogenase and its use (see, e.g., J. Biol. Chem., 276 (16): 12614-23 (2001), Japanese National Publication (Tokuhyo) No. 2002-512790, etc.) are reported. Besides the larreatricin hydroxylase gene are cloned from Larrea tridentate (see, e.g., Proc. Nat. Acad. Sci. USA, 100: 10641 (2003), etc.).
In the sesame lignan biosynthesis, it was speculated that piperitol synthase would act on pinoresinol to synthesize piperitol and in turn sesamin synthase would act on this piperitol to synthesize sesamin. However, it has become clear that cytochrome P450 cloned from S. indicum, i.e., CYP81Q1, that alone gives sesamin from pinoresinol via piperitol (WO 2005/030944; cf. FIG. 1).
In recent years, attention has been drawn not only to lignans but to methylated lignans. It is known that some of the lignan molecules described above are present in plants as glycosides. For instance, sesaminol glycosides (sesaminol 2′-O-β-D-glucopyranoside; sesaminol 2′-O-β-D-glucopyranosyl (1-2)-O-β-D-glucopyranoside; and sesaminol 2′-O-β-D-glucopyranosyl (1-2)-O-(-β-D-glucopyranosyl(1-6))-β-D-glucopyranoside)), and pinoresinol glycosides (pinoresinol 4′-O-β-D-glucopyranosyl (1-6)-β-D-glucopyranoside; pinoresinol 4′-O-β-D-glucopyranosyl (1-2)-β-D-glucopyranoside; pinoresinol 4′-O-β-D-glucopyranosyl (1-6)-O-(β-D-glucopyranosyl (1-6)) β-D-glucopyranoside; and pinoresinol di-O-β-D-glucopyranoside)), etc. are present in sesame seeds; (+)-pinoresinol 4′-O-β-D-glucoside and (−)-matairesinol-4-O-glucoside, etc. are present in Forsythia intermedia; and secolariciresinol diglucoside and pinoresinol diglucoside, etc. are present in Linum usitatissimum (see, e.g., Journal of Natural Medicines, 32, 194 (1978), Tetrahedron, 14: 649 (2003) and Phytochemistry, 58: 587 (2001)).
Pinoresinol glycosides and sesaminol glycosides contained in sesame (see, e.g., Katsuzaki, H. et al., Biosci. Biotech. Biochem., 56, 2087-2088 (1992)) show potent antioxidative properties in the water-soluble region, and are expected to yield different applications than lipophilic antioxidants (e.g., tocopherol). Also, the following mechanism of action is proposed for lignan glycosides. In lignan glycosides, the phenolic hydroxyl group, which is a functional group exhibiting antioxidative effect, is protected by sugars which themselves have but taken up into the body and then hydrolyzed by the action of β-glucosidase from enterobacteria to produce lipophilic lignans as the aglycone portion. This aglycone is absorbed into the intestines and carried to various organs via the blood to prevent oxidative damages in biomembranes of the organs, etc. Based on the action mechanism, lignan glycosides are expected to involve applications as preventive diets for arteriosclerosis (see, e.g., T. Osawa: Anticarcinogenesis and Radiation Protection 2: p. 327, Plenum Press, New York (1991)).
Methylated lignans are known as lignan derivatives other than lignan glycosides. Like lignan glycosides, methylated lignans are also lignans, which phenolic hydroxyl groups that are the functional groups relevant for antioxidative properties are blocked by methyl groups and the methoxy structure is thus assigned. It is reported that the furofuran type lignans include kobusin which is methylated piperitol in its 4-hydroxy group and sesangolin which is methylated sesaminol in its 2′ hydroxy group (cf. Phytochemistry, 47, 583-591 (1998); and J. Org. Chem., 27, 3232-3235 (1962)) (FIG. 1). However, enzymes that catalyze these synthetic reactions are unknown and so far no report has been made on purification or isolation of the enzymes for methylation of furofuran type lignans and genes encoding the same.
It is known that proteins having the particular function to catalyze transmethylation have similar amino acid sequences even in plants of different species (see, e.g., Plant Cell, 14, 505-519 (2002)).